The renal tubule regulates the absorption and secretion of water and metabolites by selective placement of lipids, ectoenzymes, receptors, channels and transporters on either the apical or basolateral surface of the epithelial cells. It is clear that (a) receptor-mediated interactions with the components of the basement membrane, (b) junctional complexes between cells, and (c) a polarized cytoskeleton are all essential for the formation and maintenance of this asymmetry. Derangement of any of these features is known to produce disease as seen for autosomal dominant polycystic kidney disease, carcinogenesis and ischemia-induced acute renal failure. In this latter case, the recovery of cell morphology is known to precede the recovery of some polarized functions due to the need to reestablish polarized expression of the mixed apical and basolateral surface lipids and proteins. However little is known about how the cell surface proteins are initially sorted within the trans-Golgi network for selective vesicular delivery to either membrane domain. While tyrosine- motifs in the cytoplasmic domain of basolateral proteins and glycolipid anchors on apical proteins, represent targeting signals for polarized expression, no such signals have been identified for apical transmembrane proteins. However, characterization of polarized cultures of LLC-PK/1 and MDCK renal epithelial cells transfected with the cDNA for the smallest of the apical estoenzymes, gammaglutamyltranspeptidase (gammaGT), indicates that the apical targeting signal(s) is within its ectodomain. In particular, preliminary studies indicate that one signal is a site of O- linked glycosylation adjacent to the membrane anchor domain, and that it affects targeting of both the transmembrane and secretory forms of the gammaGT. This is the first such detailed report of this type of apical targeting signal. Thus, the goals of this proposal are: 1. To identify specific domains and residues within the rat gammaGT involved in apical targeting. Both mutated forms of the rat gammaGT cDNA and chimeras of the rat (polarized) and E. coli (nonpolarized) gammaGTs will be prepared by recombinant DNA techniques, and the new derivatives will be expressed and characterized in polarized cultures of both MDCK (dog) and LLC-PK/1 (pig) renal epithelial cells. 2. To characterize the features of the gammaGT structure which are essential for its expression, stability and enzymatic activity. Mutant derivatives of the gammaGT will be prepared by site-specific mutagenesis of the gammaGT cDNA and these will be expressed both in Chinese Hamster Ovary cells, which lack endogenous gammaGT activity, and in polarized cultures of MDCK and LLC-PK/1 cells. Mutants are designed to identify (a) any disulfide bonds; (b) the specific sites of N-linked and O-linked glycosylation; (c) essential residues in the enzyme active site; and (d) the function of the propeptide cleavage. This detailed characterization of the structure/function relationships of the gammaGT will also enhance our basic understanding of this extremely important microvillar enzyme which hydrolyses the gamma-glutamyl bond of glutathione, thus providing a critical step in both the recovery of cysteine and the regulation of the body's oxidative/reductive state.